Methods for the formation of hydrogels using thiosulfonate compositions and uses thereof

ABSTRACT

The present invention provides both crosslinked polymer compositions capable of forming hydrogels upon exposure to an aqueous environment and thiosulfonate hydrogel-forming components. The thiosulfonate hydrogel-forming components of the invention are preferably multi-arm thiosulfonate polymer derivatives that form a crosslinked polymer composition when exposed to a base without requiring the presence of a second cross-linking reagent, redox catalyst, or radiation. Methods for forming hydrogel compositions, as well as methods for using the hydrogels, are also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/904,985, filed Sep. 27, 2007, now U.S. Pat. No. 7,598,338, which is adivisional of U.S. patent application Ser. No. 10/751,010, filed Dec.31, 2003, now U.S. Pat. No. 7,312,301, which claims the benefit ofpriority to U.S. Provisional Patent Application No. 60/437,252, filedDec. 31, 2002, all of which are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates generally to the field of polymerchemistry, and more particularly to the formation of hydrogels usingthiosulfonate derivatives of water-soluble polymers.

The hydrophilic polymer poly(ethylene glycol), abbreviated “PEG,” alsoknown as poly(ethylene oxide) abbreviated “PEO,” poly(oxyethylene)abbreviated “POE,” and poly(oxirane), is of considerable utility inbiological applications and medicine. In its most common form, PEG is alinear polymer terminated at each end with hydroxyl groups:HO—CH₂CH₂O—(CH₂CH₂O)_(n′)—CH₂CH₂—OHwherein (n′) represents the number of repeating ethylene oxide monomers.

The above polymer, α-,ω-dihydroxylpoly(ethylene glycol), can berepresented in brief form as HO-PEG-OH where it is understood that the-PEG- symbol represents the following structural unit:—CH₂CH₂O—(CH₂CH₂O)_(n′)—CH₂CH₂—where (n′) typically ranges from about 3 to about 4000.

A common form of PEG is methoxy-PEG-OH, or mPEG in brief, in which oneterminus is the relatively inert methoxy group, while the other terminusis a hydroxyl group that is subject to ready chemical modification. Thestructure of mPEG is given below:CH₃O—CH₂CH₂O—(CH₂CH₂O)_(n′)—CH₂CH₂—OHwherein (n′) typically ranges from about 3 to about 4000.

Random or block copolymers of ethylene oxide and propylene oxide, shownbelow, are closely related to PEG in their chemistry, and they can besubstituted for PEG in many of its applications.

wherein each R is independently H or CH₃ and (n′) typically ranges fromabout 3 to about 4000.

PEG is a polymer that is not only water soluble, but also is nontoxicand nonimmunogenic. Because of these properties, PEG has been covalentlyattached to insoluble molecules wherein the resulting PEG-moleculeconjugate is soluble. For example, it has been shown that thewater-insoluble drug paclitaxel, when coupled to PEG, becomes watersoluble. Greenwald et al. (1995) J. Org. Chem. 60:331-6.

PEG has also been used to form crosslinked matrices or gels. While suchPEG-formed matrices and gels are often substantially nonsoluble, theyare swellable in water. PEG hydrogels, which are water-swollen gels,have been used for wound covering and drug delivery. PEG hydrogels areprepared by incorporating PEG into a chemically crosslinked network ormatrix so that the addition of water produces an insoluble, swollen gel.One application of such hydrogels involves the delivery of drugs whereinthe drug molecules are entrapped within the crosslinked matrix. Deliveryof the drug is effected as drug molecules pass through the intersticesassociated within the matrix and ultimately leave the matrix.

One approach for preparing PEG hydrogels is described in U.S. Pat. No.4,894,238, in which hydrolytically stable and nondegradable urethane(also referred to as carbamate) linkages are described as providing ameans to connect the termini of linear polymers. For example, acrosslinked network having urethane linkages is described as beingprepared by combining PEG with a triol and a diisocyanate.

Another approach for preparing nondegradable PEG hydrogels is describedin Gayet (1996) J. Control. Release 38:177-84. In this approach, linearPEG is activated as the p-nitrophenylcarbonate and crosslinked byreaction with bovine serum albumin. Again, the linkages formed in thisapproach are hydrolytically stable urethane linkages.

U.S. Pat. No. 3,963,805 describes nondegradable PEG networks prepared byrandom entanglement of PEG chains. The described approach requires theuse of PEG with acrylic acid and a free radical initiator such as acetylperoxide.

U.S. Pat. No. 4,424,311 describes PEG hydrogels prepared bycopolymerization of PEG methacrylate with other comonomers such asmethyl methacrylate. This vinyl polymerization will produce apolyethylene backbone with PEG attached.

Sawhney et al. (1993) Macromolecules 26:581 describes the preparation ofblock copolymers of polyglycolide or polylactide and PEG that areterminated with acrylate groups. Vinyl polymerization of the acrylategroups produces an insoluble, crosslinked gel with a polyethylenebackbone. The ester groups associated with polylactide and polyglycolidesegments within the polymer backbone are susceptible to slow hydrolyticbreakdown, with the result that the crosslinked gel undergoes slowdegradation and dissolution.

Other approaches for preparing nondegradable PEG hydrogels involveradiation-induced crosslinking of high molecular weight PEGs.

These prior art methods result in the incorporation of substantialnonPEG elements into the hydrogel composition including crosslinkingagents and catalysts, and/or require the use of radiation as acrosslinking initiator. NonPEG elements, however, tend to introducecomplexity into the hydrogel. Furthermore, the presence of nonPEGelements can result in toxic components being released in vivo upon thedegradation and dissolution of the matrix. Further, harsh gellingconditions can inactivate or degrade drug substances that are oftenincorporated within a hydrogel composition.

As such, it would be desirable to provide improved hydrogel compositionsand methods for forming such hydrogel compositions that are suited forbiological applications. The present invention addresses these and otherneeds in the art by providing, inter alia, hydrogels lacking undesirablecomponents as well as methods for forming hydrogels that do not requireharsh conditions.

SUMMARY OF THE INVENTION

It is an object of the invention to provide crosslinked polymercompositions capable of forming hydrogels upon exposure to an aqueousenvironment and hydrogel-forming components. The hydrogel-formingcomponents of the invention are preferably multi-arm thiosulfonatepolymer derivatives. It has been unexpectedly found that multi-armthiosulfonate polymer derivatives form a crosslinked polymer compositionwhen exposed to a base and without requiring the presence of a secondcross-linking reagent, redox catalyst, or radiation. In one embodiment,such multi-arm thiosulfonate polymer derivatives can also form ahydrogel by reaction with polymer derivatives having at least two thiolgroups.

In one aspect of the invention, compositions are provided comprising ahydrogel-forming component of Formula (I):

wherein:

-   -   Y is a moiety derived from a molecule having at least three        nucleophilic groups;    -   POLY is a water-soluble polymer;    -   (n) is an integer from 3 to about 100,    -   X is a linking group; and    -   R is an organic radical such as an alkyl or aryl group.

Optionally, the hydrogel-forming component of Formula (I) can contain atleast one degradable linkage. A nonlimiting list of degradable linkagesinclude those selected from the group consisting of amides, esters,carbonates, acetals, orthoesters, phosphates, and thiolesters.Advantageously, the presence of one or more degradable linkages in thehydrogel-forming component allows for the degradation of the polymerchains (e.g., by hydrolysis or enzymatic degradation) of thecorresponding hydrogel. In this way, the breakdown and dissolution ofthe hydrogel can be effected following in vivo administration.

The invention further provides compositions comprising ahydrogel-forming component of Formula (II):

wherein each of Y, POLY, X, R, and (n) are as defined above with respectto Formula (I), and further wherein POLY′ is a water-soluble polymer(either the same or different than POLY), Z is a functional group havinglow reactivity (preferably no reactivity) with thiosulfonate, sulfhydryl(i.e., —SH), and disulfide linkages (i.e., —S—S— linkages), and (m) is apositive integer. Thus, the value of (m) can satisfy one or more of thefollowing ranges: 1 or more, 2 or more, 3 or more, 5 or more, 10 ormore, 15 or more, 50 or more, 75 or more, and 100 or more.

As previously indicated, POLY′ can be the same or different than POLY,POLY′ (like POLY) optionally includes a degradable linkage. With respectto the functional group “Z,” nonlimiting exemplary functional groupsfrom which Z may be selected include: active carbonate, acetal,acetamide, acrylol, aldehyde, aldehyde hydrates, alkenyl, acrylate,methacrylate, acrylamide, active sulfone, amine, carboxylic acid,epoxide, hydrazide, hydroxyl, glycol, glyoxals, guanido, isocyanate,isothiocyanate, keto, orthopyridyl-disulfide, dithiopyridine,vinylsulfone, vinylpyridine, diones, mesylates, tosylates,thiosulfonate, and tresylate and the like, as well as protected forms ofany of the foregoing. Z may also include: N-succinimidyl, succinimidylpropionate, succinimidyl succinate, succinimidyl, glycidyl ether,oxycarbonylimidazole, and p-nitrophenylcarbonate.

Yet another embodiment of the invention employs molecules of Formula(III):

wherein Y, X, R, and (n) are as defined above with respect to Formula(I), and PEG is a high or low molecular weight poly(ethylene glycol)moiety, optionally containing a degradable linkage.

In yet another aspect of the invention, hydrogels are provided. Thehydrogels comprise polymers crosslinked to each other using disulfidebonds. The hydrogels can be prepared by linking one or more of thehydrogel-forming components encompassed by any of Formulas I-III.Typically, the linking of each hydrogel-forming component is carried outby treating a composition of hydrogel-forming components with a base.Preferably, the hydrogel is formed from hydrogel-forming componentsencompassed by Formula III, wherein (n) is 4. It is believed that suchhydrogel-forming components are especially useful in the formation ofhydrogels compatible with a variety of applications including biologicalapplications.

It was unexpectedly discovered that the presently described hydrogels donot require the addition of a separate cross-linking agent or radiation(e.g., UV or microwave light) to result in their formation. Stated,differently, it was discovered that neither a separate cross-linkingagent nor radiation was required to initiate the crosslinking reactionrequired to form a hydrogel with the hydrogel-forming componentsprovided herein. As such, hydrogels of the invention can be formed fromsingle component systems that are capable of in situ gelling uponexposure to base.

In addition to being suitable for hydrogel formation in situ, thehydrogels of the invention can be molded in a variety of shapes for useas scaffolds in biological applications and tissue engineering. Themolded hydrogels are comprised of multiple layers or regions comprisingdifferent hydrogel compositions.

Another aspect of the invention provides for hydrogels formed fromhydrogel-forming components of Formulas I-III that have been furtherstabilized by crosslinking with homofunctional or heterofunctionalcrosslinking agents. Crosslinking can also be accomplished byincorporating a naturally occurring or synthetic polymer bearing two ormore groups capable of reacting with thiosulfonate groups, thiols ordisulfides to form covalent linkages.

Yet another aspect of the invention provides for hydrogel-formingcomponents and hydrogels associated with at least one biologicallyactive moiety. Association of biologically active moieties can bethrough covalent attachment, either directly to the hydrogel-formingcomponent or through the use of agents that couple the biologicallyactive moiety to the hydrogel-forming component. Alternatively or inaddition, biologically active moieties can be entrapped within thehydrogels using noncovalent interactions such as ionic interactions.

In yet another aspect of the invention, hydrogel-forming component andhydrogels associated with at least one active agent can also be used forlocalized delivery of the active agent. For example, active agents canbe delivered from the hydrogel to a local tissue site, therebyfacilitating tissue healing and regeneration.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will now be described more fully. This inventionmay, however, be embodied in many different forms and should not beconstrued as limited to the embodiments set forth herein; rather, theseembodiments are provided so that this disclosure will be thorough andcomplete, and will fully convey the scope of the invention to thoseskilled in the art

DEFINITIONS

It must be noted that, as used in this specification and the claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “adrug” includes a single drug as well as two or more of the same ordifferent drugs, reference to a “hydrogel-forming component” includes asingle hydrogel-forming component as well as two or more of the same ofdifferent hydrogel-forming components, and the like.

The terms “functional group,” “active moiety,” “activating group,”“reactive site,” “endgroup,” “chemically reactive group” and “chemicallyreactive moiety” are used in the art and herein to refer to distinct,definable portions or units of a molecule. The terms are somewhatsynonymous in the chemical arts and are used herein to indicate theportions of molecules that perform some function or activity and arereactive with other molecules. The term “active,” when used inconjunction with functional groups, is intended to include thosefunctional groups that react readily with electrophilic or nucleophilicgroups on other molecules, in contrast to those groups that requirestrong catalysts or highly impractical reaction conditions in order toreact (i.e., “nonreactive” or “inert” groups). For example, as would beunderstood in the art, the term “active ester” includes those estersthat react readily with nucleophilic groups such as amines. Exemplaryactive esters include N-hydroxysuccinimidyl esters or 1-benzotriazolylesters. Typically, an active ester will react with an amine in aqueousmedium in a matter of minutes, whereas certain esters, such as methyl orethyl esters, require a strong catalyst in order to react with anucleophilic group. As used herein, the term “functional group” includesprotected functional groups.

The term “protected functional group” refers to the presence of aprotecting group or moiety that prevents reaction of the chemicallyreactive functional group under certain reaction conditions. Theprotecting group will vary depending on the type of chemically reactivegroup being protected and the reaction conditions employed. For example,if the chemically reactive group is an amine or a hydrazide, theprotecting group can be selected from the group of tert-butyloxycarbonyl(t-Boc) and 9-fluorenylmethoxycarbonyl (Fmoc). If the chemicallyreactive group is a thiol, the protecting group can beorthopyridyldisulfide. If the chemically reactive group is a carboxylicacid, such as butanoic or propionic acid, or a hydroxyl group, theprotecting group can be benzyl or an alkyl group such as methyl, ethyl,or tert-butyl. Other protecting groups known in the art may also be usedin the invention, see for example, Greene, T. W., et al., ProtectiveGroups In Organic Synthesis, 3rd ed., John Wiley & Sons, New York, N.Y.(1999).

An “organic radical,” in the context describing a structure, astructural formula, a molecule, and so forth, refers to acarbon-containing moiety wherein a carbon atom provides a point ofattachment. Exemplary organic radicals include, alkyl (e.g., loweralkyl), substituted alkyl (including heteroalkyl, and chain-substitutedheteroalkyl), alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl,heterocyclicyl, substituted heterocyclicyl, and so forth.

“Alkyl” refers to a hydrocarbon chain, typically ranging from about 1 to15 atoms in length. Such hydrocarbon chains are preferably but notnecessarily saturated and may be branched or straight chain, althoughtypically straight chain is preferred. Exemplary alkyl groups includemethyl, ethyl, propyl, butyl, pentyl, 1-methylbutyl, 1-ethylpropyl,3-methylpentyl, and the like. As used herein, “alkyl” includescycloalkyl as well as cycloalkylene-containing alkyl.

“Lower alkyl” refers to an alkyl group containing from 1 to 6 carbonatoms, and may be straight chain or branched, as exemplified by methyl,ethyl, n-butyl, i-butyl, t-butyl.

“Cycloalkyl” refers to a saturated or unsaturated cyclic hydrocarbonchain, including bridged, fused, or spiro cyclic compounds, preferablymade up of 3 to about 12 carbon atoms, more preferably 3 to about 8.“Cycloalkylene” refers to a cycloalkyl group that is inserted into analkyl chain by bonding of the chain at any two carbons in the cyclicring system.

“Alkoxy” refers to an —O—R group, wherein R is alkyl or substitutedalkyl, preferably C₁₋₆ alkyl (e.g., methoxy, ethoxy, propyloxy, etc.).

As used herein, “alkenyl” refers to a branched or unbranched hydrocarbongroup of 2 to 15 atoms in length, containing at least one double bond,such as ethenyl, n-propenyl, isopropenyl, n-butenyl, isobutenyl,octenyl, decenyl, tetradecenyl, and the like.

The term “alkynyl” as used herein refers to branched or unbranchedhydrocarbon group of 2 to 15 atoms in length, containing at least onetriple bond, and includes, for example, ethynyl, propynyl, butynyl,octynyl, decynyl, and so forth.

“Aryl” means one or more aromatic rings, each of 5 or 6 core carbonatoms. Aryl includes multiple aryl rings that may be fused, as innaphthyl or unfused, as in biphenyl. Aryl rings may also be fused orunfused with one or more cyclic hydrocarbon, heteroaryl, or heterocyclicrings. As used herein, “aryl” includes heteroaryl.

“Heteroaryl” is an aryl group containing from one to four heteroatoms,preferably sulfur, oxygen, and nitrogen, or a combination thereof.Heteroaryl rings may also be fused with one or more cyclic hydrocarbon,heterocyclic, aryl, or heteroaryl rings.

“Heterocyclicyl” or “heterocyclic” means a group of one or more rings of5-12 atoms, preferably 5-7 atoms, with or without unsaturation oraromatic character and having at least one ring atom which is not acarbon. Preferred heteroatoms include sulfur, oxygen, and nitrogen.

The term “substituted” as in, for example, “substituted alkyl,” refersto a moiety (e.g., an alkyl group) substituted with one or morenon-interfering substituents, such as, but not limited to: C₃₋₈cycloalkyl, e.g., cyclopropyl, cyclobutyl, and the like; halo, e.g.,fluoro, chloro, bromo, and iodo; cyano; alkoxy, lower phenyl;substituted phenyl; and the like. “Substituted aryl” is aryl having oneor more noninterfering groups as a substituent. For substitutions on aphenyl ring, the substituents may be in any orientation (i.e., ortho,meta, or para).

“Substituted heteroaryl” is heteroaryl having one or more noninterferinggroups as substituents.

“Substituted heterocyclicyl” is a heterocyclicyl group having one ormore side chains formed from noninterfering substituents.

“Noninterfering substituents” are those groups that, when present in amolecule, are typically nonreactive with other functional groupscontained within the molecule.

“Electrophile” refers to an ion or atom or collection of atoms, that maybe ionic, having an electrophilic center, i.e., a center that iselectron seeking, capable of reacting with a nucleophile.

“Nucleophile” refers to an ion or atom or collection of atoms that maybe ionic having a nucleophilic center, i.e., a center that is seeking anelectrophilic center or with an electrophile.

As used herein, “nonpeptidic” refers to a structure substantially freeof amino acids connected via peptide linkages. Thus, for example, whennonpeptidic is used in reference to a polymer backbone, the polymerbackbone is substantially free of amino acids connected via peptidelinkages. The polymer backbone may, however, include a minor number ofpeptide linkages spaced along the length of the backbone, such as, forexample, no more than about 1 peptide linkage per about 50 monomerunits.

A “polymer conjugate” refers to a water-soluble polymer covalentlyattached to a biologically active moiety, as defined herein. In the casethat a polymer conjugate is reacted with a second polymer so as to forman extended polymer backbone, whether or not the joinder of the polymersis with a peptidic or other linkage, the term “polymer conjugate” refersto the overall length of polymer bound to the biologically active agent.

The term “linkage” or “linker” is used herein to refer to an atom,groups of atoms, or bonds that are normally formed as the result of achemical reaction. A linker of the invention typically links adjacentmoieties, such as two polymer segments, via one or more covalent bonds.Hydrolytically stable linkages are linkages that are substantiallystable in water and do not react to any significant degree with water atuseful pHs, e.g., under physiological conditions, for an extended periodof time, perhaps even indefinitely. A hydrolytically unstable ordegradable linkages means a linkage that is degradable in water or inaqueous solutions, including for example, blood, plasma or otherphysiological fluid. Enzymatically unstable or degradable linkagesencompass those linkages can be degraded by one or more enzymes.

The terms “drug,” “biologically active molecule,” “biologically activemoiety,” “biologically active agent,” “active agent,” and the like meanany substance which can affect any physical or biochemical properties ofa biological organism, including but not limited to viruses, bacteria,fungi, plants, animals, and humans. In particular, as used herein,biologically active molecules include any substance intended fordiagnosis, cure, mitigation, treatment, or prevention of disease inhumans or other animals, or to otherwise enhance physical or mentalwell-being of humans or animals. Examples of biologically activemolecules include, but are not limited to, peptides, proteins, enzymes,small molecule drugs (e.g., nonpeptidic drugs), dyes, lipids,nucleosides, oligonucleotides, polynucleotides, nucleic acids, cells,viruses, liposomes, microparticles and micelles. Classes of biologicallyactive agents that are suitable for use with the invention include, butare not limited to, antibiotics, fungicides, anti-viral agents,anti-inflammatory agents, anti-tumor agents, cardiovascular agents,anti-anxiety agents, hormones, growth factors, steroidal agents, and thelike.

The terms “low weight polymer” and “low molecular weight polymer”broadly refer to a linear, branched, multi-arm, or forked polymerbackbones comprising a water-soluble and nonpeptidic polymer having from1 to about 120 repeating units. These polymers typically have from 1 to2 functional groups, typically located at opposite termini on a linearpolymer, to about 300, which can be located at the termini of highlybranched or multiarmed structures, although a smaller number may belocated along the polymer backbone. Although the molecular weight of thesmall polymer or oligomer can vary, it is typically in the range of fromabout 100 Da to about 10,000 Da, depending, of course, on the molecularweight of the individual repeating units. In the case of PEG, one PEGmonomer unit has a molecular weight of about 44 Da and low weightpolymers will have a molecular weight of from about 44 Da to about 5280Da. Molecular weights of 2000, 3200, 3400, and 5,000 are availablecommercially.

The terms “high weight polymer” and “high molecular weight polymer”broadly refer to a linear, branched, or multi-arm polymer backbonecomprising a water-soluble and nonpeptidic polymer having more thanabout 200 repeating units. These polymers typically have from 1 to 2functional groups, typically located at opposite termini on a linearpolymer, to about 300, which can be located along the polymer backboneor at the termini of highly branched or multiarmed structures. Forkedstructures are also contemplated in which a terminus is branched toprovide two functionalities. In the case of PEG, high weight polymershave a molecular weight above about 8,800 Da. Commercially availablePEGs include those having a nominal molecular weight of 10,000 Da,12,000 Da, 15,000 Da, 18,000 Da, 20,000 Da, 30,000 Da, 40,000 Da, andabove. Straight and branched PEGs are readily available at highermolecular weights.

Reference to a “molecular weight” in the context of a water-solublepolymer refers to the mass average molecular weight of the polymer,typically determined by size exclusion chromatography, light scatteringtechniques, or intrinsic velocity determination in1,2,4-trichlorobenzene.

As used herein, “PEG” broadly refers to a linear, multi-arm, or branchedpolymer backbone comprising a water-soluble and non-peptidic polymerhaving repeat CH₂CH₂O units. The polymer α,ω-dihydroxypoly(ethyleneglycol), can be represented in brief form as HO-PEG-OH where it isunderstood that the -PEG- symbol represents the following structuralunit —CH₂CH₂O—(CH₂CH₂O)_(n′)—CH₂CH₂— where (n′) typically ranges fromabout 3 to about 4000. The PEG family of polymers generally exhibits theproperties of solubility in water and in many organic solvents, lack oftoxicity, and lack of immunogenicity. The term PEG should be understoodto be inclusive and to include poly(ethylene glycol) in any of itslinear, branched or multi-arm forms, including alkoxy PEG, bifunctionalPEG, forked PEG, branched PEG, pendant PEG, and PEG with degradablelinkages therein.

PEG, in any of the forms described herein, is typically clear,colorless, odorless, soluble in water, stable to heat, inert to manychemical agents, does not hydrolyze or deteriorate (unless specificallydesigned to do so), and is generally nontoxic. Poly(ethylene glycol) isconsidered to be biocompatible, which is to say that PEG is capable ofcoexistence with living tissues or organisms without causing harm. Morespecifically, PEG is substantially nonimmunogenic, which is to say thatPEG does not tend to produce an immune response in a patient. Whenattached to a molecule having some desirable function in the body, suchas a biologically active agent, the PEG tends to mask the agent and canreduce or eliminate any immune response so that an organism can toleratethe presence of the agent. PEG-containing conjugates and hydrogels tendnot to produce a substantial immune response or cause clotting or otherundesirable effects.

As used herein, “hydrogels” are compositions resulting from theassociation of one or more types of molecules to form a substantiallynonwater soluble material. “Hydrogel” is not meant to indicate that thematerial is rigid, but rather that the composition has undergone linkingsuch that the components of the hydrogel substantially interact.Hydrogels of this invention will span the range of viscosity fromsolutions, some of which appear viscous in nature, through gels, whichsubstantially retain their shape and structure when unsupported.

The term “patient,” refers to a living organism suffering from or proneto a condition that can be prevented or treated by administration of adrug, and includes both humans and animals.

“Optional” or “optionally” means that the subsequently describedcircumstance may or may not occur, so that the description includesinstances where the circumstance occurs and instances where it does not.

II. Thiosulfonate Polymer Derivatives

In a first aspect of the invention, thiosulfonate polymer derivativesare provided which are capable of crosslinking upon exposure to basicconditions. Preferably, the thiosulfonate polymer derivatives of theinvention are multi-arm thiosulfonates of water-soluble polymers. In apreferred embodiment of the invention, hydrogel-forming components areprovided comprising molecules encompassed by Formula (I):

wherein:

-   -   Y is a moiety derived from a molecule having at least three        nucleophilic groups;    -   POLY is a water-soluble polymer;    -   (n) is an integer from 3 to about 100;    -   X is a linking group; and    -   R is an organic radical such as an alkyl or aryl group.

The water-soluble polymer (such as POLY and POLY′) useful in thisinvention include any water-soluble polymers and the invention is notlimited in this regard. The water-soluble polymer can include polyamidessuch as polypeptides. In addition, water-soluble polymers that arenonpeptidic are particularly useful in the invention. By way of example,a water-soluble polymer as used herein can be poly(alkylene glycol),including poly(ethylene glycol) (PEG), poly(propylene glycol) (PPG) orcopolymers of poly(alkylene) glycols including copolymers ofpoly(ethylene glycol) and poly(propylene glycol), poly(lactide),poly(glycolide), poly(caprolactone), poly(oxyethylated polyol),poly(olefinic alcohol), poly(vinylpyrrolidone),N-(2-hydroypropyl)methacrylamide, poly-1,3-dioxolane,poly-1,3,6-trioxolane, poly(hydroxypropylmethacrylamide),poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate),poly(α-hydroxy acid), poly(vinyl alcohol), polyphosphazene,polyoxazoline, poly(saccharides), carboxymethylcellulose, dextran,copolymers of ethylene/maleic anhydride copolymers,polylactide/polyglycolide copolymers, propylene oxide/ethylene oxidecopolymers, copolymers of polyethylene glycol and an and amino acid, andpoly(N-acryloylmorpholine), such as described in U.S. Pat. No.5,629,384, which is incorporated by reference herein in its entirety.The preferred polymer is poly(ethylene glycol) having a size from about200 Da to about 20,000 Da, or more preferably from about 500 Da to about15,000 Da or more preferably from about 600 Da to about 6000 Da.

Those of ordinary skill in the art will recognize that the foregoinglist for substantially water soluble polymers is by no means exhaustiveand is merely illustrative, and that all polymeric materials having thequalities described above are contemplated.

The linking group serves to link the thiosulfonate to the water-solublepolymer. Preferred linking groups are selected from the group consistingof alkylene groups, alkylene amides, alkylene esters, or alkyleneethers. Specific examples of a suitable linking group include thoseselected from the group consisting of —O—, —S—, —C(O)—, —O—C(O)—,—C(O)—O—, —C(O)—NH—, —NH—C(O)—NH—, —O—C(O)—NH—, —C(S)—, —CH₂—,—CH₂—CH₂—, —CH₂—CH₂—CH₂—, —CH₂—CH₂—CH₂—CH₂—, —O—CH₂—, —CH₂—O—,—O—CH₂—CH₂—, —CH₂—O—CH₂—, —CH₂—CH₂—O—, —O—CH₂—CH₂—CH₂—, —CH₂—O—CH₂—CH₂—,—CH₂—CH₂—O—CH₂—, —CH₂—CH₂—CH₂—O—, —O—CH₂—CH₂—CH₂—CH₂—,—CH₂—O—CH₂—CH₂—CH₂—, —CH₂—CH₂—O—CH₂—CH₂—, —CH₂—CH₂—CH₂—O—CH₂—,—CH₂—CH₂—CH₂—CH₂—O—, —C(O)—NH—CH₂—, —C(O)—NH—CH₂—CH₂—,—CH₂—C(O)—NH—CH₂—, —CH₂—CH₂—C(O)—NH—, —C(O)—NH—CH₂—CH₂—CH₂—,—CH₂—C(O)—NH—CH₂—CH₂—, —CH₂—CH₂—C(O)—NH—CH₂—, —CH₂—CH₂—CH₂—C(O)—NH—,—C(O)—NH—CH₂—CH₂—CH₂—CH₂—, —CH₂—C(O)—NH—CH₂—CH₂—CH₂—,—CH₂—CH₂—C(O)—NH—CH₂—CH₂—, —CH₂—CH₂—CH₂—C(O)—NH—CH₂—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—, —CH₂—CH₂—CH₂—CH₂—C(O)—NH—, —C(O)—O—CH₂—,—CH₂—C(O)—O—CH₂—, —CH₂—CH₂—C(O)—O—CH₂—, —C(O)—O—CH₂—CH₂—, —NH—C(O)—CH₂—,—CH₂—NH—C(O)—CH₂—, —CH₂—CH₂—NH—C(O)—CH₂—, —NH—C(O)—CH₂—CH₂—,—CH₂—NH—C(O)—CH₂—CH₂—, —CH₂—CH₂—NH—C(O)—CH₂—CH₂—, —C(O)—NH—CH₂—,—C(O)—NH—CH₂—CH₂—, —O—C(O)—NH—CH₂—, —O—C(O)—NH—CH₂—CH₂—,—O—C(O)—NH—CH₂—CH₂—CH₂—, —NH—CH₂—, —NH—CH₂—CH₂—, —CH₂—NH—CH₂—,—CH₂—CH₂—NH—CH₂—, —C(O)—CH₂—, —C(O)—CH₂—CH₂—, —CH₂—C(O)—CH₂—,—CH₂—CH₂—C(O)—CH₂—, —CH₂—CH₂—C(O)—CH₂—CH₂—, —CH₂—CH₂—C(O)—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—, —CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—CH₂—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—CH₂—CH₂—,—O—C(O)—NH—[CH₂]₀₋₆—(OCH₂CH₂)₀₋₂—, —C(O)—NH—(CH₂)₁₋₆—NH—C(O)—,—NH—C(O)—NH—(CH₂)₁₋₆—NH—C(O)—, —O—C(O)—CH₂—, —O—C(O)—CH₂—CH₂—, and—O—C(O)—CH₂—CH₂—CH₂—. The linking group can be joined to thewater-soluble polymer by a variety of linkages, including but notlimited to: ether, thioether, amine, amide, ester or single, double ortriple carbon-carbon bonds.

Y can be any moiety derived from a molecule having 3 or morenucleophilic groups. Preferably, the “Y” moiety has a branching corestructure providing from 3 to about 100 available groups for theattachment of POLY, and typically provides about 3 to 20, 4 to 25, or 5to 30 available groups, such that the branched polymer structure hasfrom 3 to about 100 polymer chains, although there may be more groupspresent on Y than polymer chains attached thereto. Preferred availablegroups for the attachment of polymers to Y include hydroxyl and aminogroups. Y moieties can be derived from glycerol, oligoglycerols,pentaerythritol, carbohydrates, cyclodextrin, or amine analogues ofthese molecules (e.g., Y may be a substituted glycerol or1,2,3-propane-triamine).

The value of (n) is preferably at least 3, 4, 5, 6, 10, 15, 25, 50, 75,or 100. The value of (n) depends on the nature of the Y moiety employed.Typically, however the value of (n) will satisfy one or more of thefollowing ranges: 3-4; 3-5; 3-10; 4-5; 4-6; 4-10; 5-6; 5-10; 5-25;10-25; 25-50; 50-75; and 75-100.

Optionally, the water-soluble polymer can include at least onedegradable linkage. Preferred degradable linkages are selected from thegroup consisting of amides, imines, esters, carbonates, hydrazone,acetals, orthoesters, phosphates, and thiolesters. These and otherdegradable linkages allow for the degradation of the water-solublepolymers associated therein. Degradation of water-soluble polymers, inturn, can result in the breakdown and dissolution of any hydrogel formedby degradable linkage-containing water-soluble polymers and derivativesthereof. Such linkages are typically subject to degradation byhydrolysis or enzymatic degradation.

The formation of such linkages is known to those of ordinary skill inthe art and can be accomplished by reacting two water-soluble polymers,each bearing a different reactive group such that the bond resultingfrom the two different reactive groups is a degradable linkage. Briefly,exemplary linkages can be formed as followed: imine linkages result, forexample, from reaction of an amine and an aldehyde (see, e.g., Ouchi etal. (1997) Polymer Preprints 38(1):582-3, which is incorporated hereinby reference); phosphate ester linkages are formed, for example, byreacting an alcohol with a phosphate group; hydrazone linkages result,for example, by reaction of a hydrazide and an aldehyde; acetal linkagesare formed by, for example, reaction between an aldehyde and an alcohol;orthoester linkages are formed by, for example, reaction between aformate and an alcohol. Water-soluble polymers bearing these and otherreactive groups are commercially available or can be synthesized by onehaving ordinary skill in the art.

In addition to the incorporation of degradable linkages into thewater-soluble polymer, the degradable linkages of the backbone can beemployed to covalently attach biologically active moieties to thepolymer backbones through a weak or degradable linkage moiety. Suchlinkage moieties generally degrade under physiological conditions,typically by hydrolysis or enzymatic cleavage, resulting in the releaseof the biologically active moieties from the polymer backbone.

The invention also provides molecules encompassed by Formula (II).Essentially, the molecules of Formula (II) comprise the additionalmoiety —POLY′-Z attached to the Y moiety of Formula (I). Thus, themolecules of Formula (II) comprise the following structure:

wherein each of Y, POLY, X, R, and (n) are as defined above with respectto Formula (I), and further wherein POLY′ is a water-soluble polymer(either the same or different than POLY), Z is a functional group havinglow reactivity (preferably no reactivity) with thiosulfonate, sulfhydryl(i.e., —SH), and disulfide linkages (i.e., —S—S— linkages), and (m) is apositive integer.

The value of (m) is 1 or more, 2 or more, 3 or more, 5 or more, 10 ormore, 15 or more, 25 or more, 50 or more, 75 or more, or 100 or more. Inthis embodiment, POLY′ may be the same or different than POLY. Both POLYand POLY′ are selected from the group defined for POLY in formula (I)above and may include a degradable linkage.

Z can be any functional group with a desired functionality, as would berecognized by one skilled in the art. In one embodiment, Z may be afunctional group which is of low reactivity or nonreactive withthiosulfonate, —SH, or —S—S— linkages. In addition, Z can be a groupcapable of reacting with an active agent, thereby providing ahydrogel-forming component—as well as a hydrogel formedtherefrom—containing a covalently bond active agent. Exemplaryfunctional groups in this regard include hydroxyl, active ester (e.g.N-hydroxysuccinimidyl ester or 1-benzotriazolyl ester), active carbonate(e.g. N-hydroxysuccinimidyl carbonate and 1-benzotriazolyl carbonate),acetal, aldehyde, aldehyde hydrate, alkenyl, acrylate, methacrylate,acrylamide, active sulfone, amine, hydrazide, thiol, carboxylic acid,isocyanate, isothiocyanate, maleimide, vinylsulfone, dithiopyridine,vinylpyridine, iodoacetamide, epoxide, glyoxal, dione, mesylate,tosylate, or tresylate.

Specific examples of “Z” functional groups for covalent attached to anactive agent include N-succinimidyl carbonate (see e.g., U.S. Pat. Nos.5,281,698, and 5,468,478), amine (see, e.g., Buckmann et al. (1981)Makromol. Chem. 182:1379, and Zalipsky et al. (1983) Eur. Polym. J.19:1177), hydrazide (See, e.g., Andresz et al. (1978) Makromol. Chem.179:301), succinimidyl propionate and succinimidyl butanoate (see, e.g.,Olson et al. in Poly(ethylene glycol) Chemistry & BiologicalApplications, pp 170-181, Harris & Zalipsky Eds., ACS, Washington, D.C.,1997, and U.S. Pat. No. 5,672,662), succinimidyl succinate (see, e.g.,Abuchowski et al. (1984) Cancer Biochem. Biophys. 7:175, and Joppich etal. (1979) Makromol. Chem. 180:1381), succinimidyl ester (see, e.g.,U.S. Pat. No. 4,670,417), benzotriazole carbonate (see, e.g., U.S. Pat.No. 5,650,234), glycidyl ether (see, e.g., Pitha et al. (1979) Eur. J.Biochem. 94:11, and Elling et al. (1991) Biotech. Appl. Biochem.13:354), oxycarbonylimidazole (see, e.g., Beauchamp et al. (1983) Anal.Biochem. 131:25, and Tondelli et al. (1985) J. Controlled Release1:251), p-nitrophenylcarbonate (see, e.g., Veronese, et al. (1985) Appl.Biochem. Biotech. 11:141, and Sartore et al. (1991) Appl. Biochem.Biotech. 27:45), aldehyde (see, e.g., Harris et al. (1984) J. Polym.Sci. Chem. Ed. 22:341, and U.S. Pat. Nos. 5,824,784, and 5,252,714),maleimide (see, e.g., Goodson et al. (1990) Bio/Technology 8:343, Romaniet al. (1984) Chemistry of Peptides and Proteins 2:29), and Kogan (1992)Synthetic Comm. 22:2417), orthopyridyl-disulfide (see, e.g., Woghiren etal. (1993) Bioconj. Chem. 4:314), acrylol (see, e.g., Sawhney et al.(1993) Macromolecules 26:581), and vinylsulfone (see, e.g., U.S. Pat.No. 5,900,461). All of the above references are incorporated herein byreference.

Inclusion of —POLY′-Z moieties permits a change in the characteristicsof the resultant hydrogel compositions. For example, when Z is amine,the resultant hydrogel will be cationically charged at neutral pH, andas such may form associations with negatively charged active agents suchas typical nonsteroidal anti-inflammatory drugs (“NSAIDs”), orpolyanionic materials such as condroitin sulfate, either of which maybeincorporated into the hydrogel when it is formed or after it has beenprepared. In addition, it is possible to form covalent attachments to Zgroups such as amines either directly, or through the use ofamine-reactive crosslinking agents.

Yet another embodiment of the invention employs molecules of Formula(III):

wherein Y, X, R, and (n) are as defined above with respect to Formula(I), and PEG is a high or low molecular weight poly(ethylene glycol)moiety, optionally containing a degradable linkage.

A preferred embodiment of the invention employs molecules of Formula(III), wherein (n) is 3, 4, or 8. Such 3-, 4-, and 8-arm PEG moleculesare especially useful in the formation of hydrogels compatible with avariety of applications including biological applications.

Without intending to be limited by theory, it is believed that thethiosulfonate polymer derivatives of the invention release a sulfonateleaving group upon exposure to a base, thereby generating a reactivethiol group in situ. This thiol group is then available for reactionwith appropriate electrophilic functional groups such as a thiosulfonategroup associated with the thiosulfonate-containing hydrogel-formingcomponents described herein.

Hydrogel Compositions

Another aspect of the invention is directed to crosslinked polymercompositions capable of forming a hydrogel composition upon exposure toan aqueous environment. The hydrogel compositions are characterized ashaving a relatively low gel time with a viscosity suited foradministration to a patient. The hydrogel compositions of the inventioncan be formed from the hydrogel-forming components previously described.Thus, the hydrogel-forming components are preferably thiosulfonatepolymer derivatives, and more preferably multi-arm thiosulfonate estersof a water-soluble polymer such as those encompassed by Formulas I-III.In a preferred embodiment, hydrogel compositions of the invention areformed from a composition comprising only one type of hydrogel-formingcomponent. Such a “single-component” system is advantageously lesscostly to make, easier to store and package, and relatively simple toform into a hydrogel composition. The single-component compositioncomprises a thiosulfonate polymer derivative. Moreover, the hydrogelcomposition preferably lacks by-products of crosslinking agents. Inaddition, the hydrogel composition preferably lacks redox catalysts.

As used herein, a single component hydrogel-forming compositiongenerally refers to a composition comprising only a single componentthat is involved in crosslinking to form the hydrogel composition. Assuch, a single component hydrogel-forming composition can compriseadditional components such as buffers and biologically active moieties,but will generally only include a single hydrogel-forming componentcapable of crosslinking. In some embodiments, however, the hydrogels maybe subsequently modified by the incorporation of additional componentssuch as biologically active moieties or stabilizing crosslinking agents.Modification of hydrogels to incorporate additional components may beaccomplished either by covalent or noncovalent association with thehydrogel.

Noncovalent association of additional components with the hydrogels ofthis invention may be accomplished by entrapment of the componentswithin the hydrogel during its formation. Alternatively, additionalcomponents may be associated with the hydrogels of this invention bycontacting the component with the formed hydrogel. Noncovalentassociation of active agents that are entrapped or contacted with thehydrogels of this invention may further include the formation ofcomplexes resulting from ionic interactions. In some embodiments of thisinvention, such ionic interactions can be effected by the incorporationof free amino groups into the molecules of Formula (II) where Z is anamino group. In yet other embodiments of this invention, ionicinteractions are effected by incorporating carboxyl groups into themolecules of Formula (II) where Z is a carboxyl group.

Covalent attachment of additional components to the hydrogelcompositions of the invention can be accomplished by modification of thethiosulfonate hydrogel-forming components prior to hydrogel formation,concurrent with hydrogel formation, or by modification of a hydrogelonce it has been formed. Such covalent attachment can be achievedthrough direct covalent linkage of the molecules to the hydrogel orhydrogel-forming component. By way of example, direct covalent linkageis possible where the additional component bears a pendant reactivegroup that can form a linkage with thiosulfonate, thiol or disulfidegroups present in the hydrogel-forming component or the formed hydrogel.Alternatively, additional components may be covalently attached to thehydrogel or hydrogel-forming component by the interposition of acrosslinking agent between the polymer and the additional component.Crosslinking agents useful for this purpose include homobifunctional,heterobifunctional, and trifunctional crosslinking agents.

Without intending to be limited by theory, the hydrogel compositions ofthe invention will generally comprise disulfide bonds formed betweenthiosulfonate functional groups of the hydrogel-forming components andthiol groups that are generated in situ upon exposure of thehydrogel-forming components to base. Such disulfide bonds are generallynot biodegradable unless internalized by a cell or exposed to disulfidereduction agents such as reduced gluthathione. However, hydrolyticallyor enzymatically unstable linkages can be incorporated into the polymerbackbone of the hydrogel-forming components to allow for biodegradablehydrogel compositions.

As discussed above, hydrogel compositions of the invention can thereforebe formed from single component hydrogel-forming compositions withoutthe need for a second reagent such as a crosslinking agent or redoxcatalyst. The hydrogel composition can additionally contain leavinggroups such as methane sulfonate and methane sulfinate, but ispreferably lacking crosslinking groups. Since the mixture can be formedfrom a single component composition, the end user does not have toadjust the proportion of hydrogel-forming components. This, in turn,leads to greater uniformity in the resulting hydrogel and ease of use.Further, the base crosslinking initiator is easily neutralized in vivo,thereby improving the biocompatibility of the hydrogel.

In a preferred embodiment, the hydrogel compositions of the inventionmay optionally be associated with at least one active agent, eitherthrough a covalent attachment or through a noncovalent attachment. Suchactive agents can be noncovalently associated with the hydrogelcompositions of the invention by adding them to the hydrogel-formingcomponents prior to the formation of the hydrogel, thereby entrappingthem within the gel upon its formation. Active agents can also benoncovalently associated with the hydrogels of this invention bycontacting the formed hydrogel with the active agent for a period oftime sufficient to permit incorporation within the hydrogel (e.g., aperiod of from several minutes to several hours or days).

In addition, an active agent can be incorporated into the hydrogelcompositions of the invention by covalently binding the active agentdirectly or indirectly to the functional groups present on ahydrogel-forming component (e.g., the functional group “Z” includedencompassed by a polymer of Formula II). As discussed above, theinteraction of active agents with the hydrogel compositions may bemodified through incorporation of one or more charged groups into thehydrogel forming components, or by incorporating charged components intothe hydrogel compositions themselves. Further, association of activeagents can be limited to outer surfaces or to regions accessible bydiffusion, if desired.

Any active agent can be associated with the hydrogels andhydrogel-forming components and the invention is not limited in thisregard. Suitable agents can be selected from, for example, hypnotics andsedatives, psychic energizers, tranquilizers, respiratory drugs,anticonvulsants, muscle relaxants, antiparkinson agents (dopamineantagnonists), analgesics, anti-inflammatories, antianxiety drugs(anxiolytics), appetite suppressants, antimigraine agents, musclecontractants, anti-infectives (antibiotics, antivirals, antifungals,vaccines) antiarthritics, antimalarials, antiemetics, anepileptics,bronchodilators, cytokines, growth factors, anti-cancer agents,antithrombotic agents, antihypertensives, cardiovascular drugs,antiarrhythmics, antioxicants, anti-asthma agents, hormonal agentsincluding contraceptives, sympathomimetics, diuretics, lipid regulatingagents, antiandrogenic agents, antiparasitics, anticoagulants,neoplastics, antineoplastics, hypoglycemics, nutritional agents andsupplements, growth supplements, antienteritis agents, vaccines,antibodies, diagnostic agents, and contrasting agents.

More particularly, the active agent may fall into one of a number ofstructural classes, including but not limited to small molecules(preferably insoluble small molecules), peptides, polypeptides,proteins, antibodies, antibody fragments, polysaccharides, steroids,nucleotides, oligonucleotides, polynucleotides, fats, electrolytes, andthe like. Preferably, an active agent for coupling to a polymer asdescribed herein possesses a native amino group, or alternatively, ismodified to contain at least one reactive amino group suitable forconjugating to a polymer described herein.

Specific examples of active agents suitable for covalent attachmentinclude but are not limited to aspariginase, amdoxovir (DAPD), antide,becaplermin, calcitonins, cyanovirin, denileukin diftitox,erythropoietin (EPO), EPO agonists (e.g., peptides from about 10-40amino acids in length and comprising a particular core sequence asdescribed in WO 96/40749), dornase alpha, erythropoiesis stimulatingprotein (NESP), coagulation factors such as Factor V, Factor VII, FactorVIIa, Factor VIII, Factor IX, Factor X, Factor XII, Factor XIII, vonWillebrand factor; ceredase, cerezyme, alpha-glucosidase, collagen,cyclosporin, alpha defensins, beta defensins, exedin-4, granulocytecolony stimulating factor (GCSF), thrombopoietin (TPO), alpha-1proteinase inhibitor, elcatonin, granulocyte macrophage colonystimulating factor (GMCSF), fibrinogen, filgrastim, growth hormoneshuman growth hormone (hGH), growth hormone releasing hormone (GHRH),GRO-beta, GRO-beta antibody, bone morphogenic proteins such as bonemorphogenic protein-2, bone morphogenic protein-6, OP-1; acidicfibroblast growth factor, basic fibroblast growth factor, CD-40 ligand,heparin, human serum albumin, low molecular weight heparin (LMWH),interferons such as interferon alpha, interferon beta, interferon gamma,interferon omega, interferon tau, consensus interferon; interleukins andinterleukin receptors such as interleukin-1 receptor, interleukin-2,interleukin-2 fusion proteins, interleukin-1 receptor antagonist,interleukin-3, interleukin-4, interleukin-4 receptor, interleukin-6,interleukin-8, interleukin-12, interleukin-13 receptor, interleukin-17receptor; lactoferrin and lactoferrin fragments, luteinizing hormonereleasing hormone (LHRH), insulin, pro-insulin, insulin analogues (e.g.,mono-acylated insulin as described in U.S. Pat. No. 5,922,675), amylin,C-peptide, somatostatin, somatostatin analogs including octreotide,vasopressin, follicle stimulating hormone (FSH), influenza vaccine,insulin-like growth factor (IF), insulintropin, macrophage colonystimulating factor (M-CSF), plasminogen activators such as alteplase,urokinase, reteplase, streptokinase, pamiteplase, lanoteplase, andteneteplase; nerve growth factor (NGF), osteoprotegerin,platelet-derived growth factor, tissue growth factors, transforminggrowth factor-1, vascular endothelial growth factor, leukemia inhibitingfactor, keratinocyte growth factor (KGF), glial growth factor (GGF), TCell receptors, CD molecules/antigens, tumor necrosis factor (TNF),monocyte chemoattractant protein-1, endothelial growth factors,parathyroid hormone (PTH), glucagon-like peptide, somatotropin, thymosinalpha 1, rasburicase, thymosin alpha 1 IIb/IIIa inhibitor, thymosin beta10, thymosin beta 9, thymosin beta 4, alpha-1 antitrypsin,phosphodiesterase (PDE) compounds, VLA-4 (very late antigen-4), VLA-4inhibitors, bisphosphonates, respiratory syncytial virus antibody,cystic fibrosis transmembrane regulator (CFTR) gene, deoxyreibonuclease(Dnase), bactericidal/permeability increasing protein (BPI), andanti-CMV antibody. Exemplary monoclonal antibodies include etanercept (adimeric fusion protein consisting of the extracellular ligand-bindingportion of the human 75 kD TNF receptor linked to the Fc portion ofIgG1), abciximab, adalimumab, afelimomab, alemtuzumab, antibody toB-lymphocyte, atlizumab, basiliximab, bevacizumab, biciromab,bertilimumab, CDP-571, CDP-860, CDP-870, cetuximab, clenoliximab,daclizumab, eculizumab, edrecolomab, efalizumab, epratuzumab,fontolizumab, gavilimomab, gemtuzumab ozogamicin, ibritumomab tiuxetan,infliximab, inolimomab, keliximab, labetuzumab, lerdelimumab, olizumab,radiolabeled lym-1, metelimumab, mepolizumab, mitumomab, muromonad-CD3,nebacumab, natalizumab, odulimomab, omalizumab, oregovomab, palivizumab,pemtumomab, pexelizumab, rhuMAb-VEGF, rituximab, satumomab pendetide,sevirumab, siplizumab, tositumomab, I¹³¹tositumomab, trastuzumab,tuvirumab, visilizumab, tacrine, memantine, rivastigmine, galantamine,donepezil, levetiracetam, repaglinide, atorvastatin, alefacept,vardenafil, sildenafil, and valacyclovir,

Additional agents suitable for covalent attachment include, but are notlimited to, adefovir, alosetron, amifostine, amiodarone, aminocaproicacid, aminohippurate sodium, aminoglutethimide, aminolevulinic acid,aminosalicylic acid, amsacrine, anagrelide, anastrozole, aripiprazole,asparaginase, anthracyclines, bexarotene, bicalutamide, bleomycin,buserelin, busulfan, cabergoline, capecitabine, carboplatin, carmustine,chlorambucin, cilastatin sodium, cisplatin, cladribine, clodronate,cyclophosphamide, cyproterone, cytarabine, camptothecins, 13-cisretinoic acid, all trans retinoic acid; dacarbazine, dactinomycin,daunorubicin, deferoxamine, dexamethasone, diclofenac,diethylstilbestrol, docetaxel, doxorubicin, dutasteride, epirubicin,estramustine, etoposide, exemestane, ezetimibe, fexofenadine,fludarabine, fludrocortisone, fluorouracil, fluoxymesterone, flutamide,fondaparinux, fulvestrant, gamma-hydroxybutyrate, gemcitabine,epinephrine, L-Dopa, hydroxyurea, idarubicin, ifosfamide, imatinib,irinotecan, itraconazole, goserelin, letrozole, leucovorin, levamisole,lisinopril, lovothyroxine sodium, lomustine, mechlorethamine,medroxyprogesterone, megestrol, melphalan, mercaptopurine, metaraminolbitartrate, methotrexate, metoclopramide, mexiletine, mitomycin,mitotane, mitoxantrone, naloxone, nicotine, nilutamide, nitisinone,octreotide, oxaliplatin, pamidronate, pentostatin, pilcamycin, porfimer,prednisone, procarbazine, prochlorperazine, ondansetron, palonosetron,oxaliplatin, raltitrexed, sirolimus, streptozocin, tacrolimus,pimecrolimus, tamoxifen, tegaserod, temozolomide, teniposide,testosterone, tetrahydrocannabinol, thalidomide, thioguanine, thiotepa,topotecan, treprostinil, tretinoin, valdecoxib, celecoxib, rofecoxib,valrubicin, vinblastine, vincristine, vindesine, vinorelbine,voriconazole, dolasetron, granisetron; formoterol, fluticasone,leuprolide, midazolam, alprazolam, amphotericin B, podophylotoxins,nucleoside antivirals, aroyl hydrazones, sumatriptan, eletriptan,macrolides such as erythromycin, oleandomycin, troleandomycin,roxithromycin, clarithromycin, davercin, azithromycin, flurithromycin,dirithromycin, josamycin, spiromycin, midecamycin, loratadine,desloratadine, leucomycin, miocamycin, rokitamycin, andazithromycin, andswinolide A; fluoroquinolones such as ciprofloxacin, ofloxacin,levofloxacin, trovafloxacin, alatrofloxacin, moxifloxicin, norfloxacin,enoxacin, grepafloxacin, gatifloxacin, lomefloxacin, sparfloxacin,temafloxacin, pefloxacin, amifloxacin, fleroxacin, tosufloxacin,prulifloxacin, irloxacin, pazufloxacin, clinafloxacin, and sitafloxacin;aminoglycosides such as gentamicin, netilmicin, paramecin, tobramycin,amikacin, kanamycin, neomycin, and streptomycin, vancomycin,teicoplanin, rampolanin, mideplanin, colistin, daptomycin, gramicidin,colistimethate; polymixins such as polymixin B, capreomycin, bacitracin,penems; penicillins including penicllinase-sensitive agents likepenicillin G, penicillin V; penicllinase-resistant agents likemethicillin, oxacillin, cloxacillin, dicloxacillin, floxacillin,nafcillin; gram negative microorganism active agents like ampicillin,amoxicillin, and hetacillin, cillin, and galampicillin; antipseudomonalpenicillins like carbenicillin, ticarcillin, azlocillin, mezlocillin,and piperacillin; cephalosporins like cefpodoxime, cefprozil,cefitbuten, ceftizoxime, ceftriaxone, cephalothin, cephapirin,cephalexin, cephradrine, cefoxitin, cefamandole, cefazolin,cephaloridine, cefaclor, cefadroxil, cephaloglycin, cefaroxime,ceforanide, cefotaxime, cefatrizine, cephacetrile, cefepime, cefixime,cefonicid, cefoperazone, cefotetan, cefmetazole, ceftazidime,loracarbef, and moxalactam, monobactams like aztreonam; and carbapenemssuch as imipenem, meropenem, and ertapenem, pentamidine isetionate,albuterol sulfate, lidocaine, metaproterenol sulfate, beclomethasonediprepionate, triamcinolone acetamide, budesonide acetonide,fluticasone, ipratropium bromide, flunisolide, cromolyn sodium, andergotamine tartrate; taxanes such as paclitaxel; SN-38, andtyrphostines.

Preferred small molecules for coupling to a polymer as described hereinare those having at least one naturally occurring amino group. Preferredmolecules such as these include aminohippurate sodium, amphotericin B,doxorubicin, aminocaproic acid, aminolevulinic acid, aminosalicylicacid, metaraminol bitartrate, pamidronate disodium, daunorubicin,levothyroxine sodium, lisinopril, cilastatin sodium, mexiletine,cephalexin, deferoxamine, and amifostine.

Preferred peptides or proteins for coupling to a polymer as describedherein include EPO, IFN-α, IFN-β, consensus IFN, Factor VIII, Factor IX,GCSF, GMCSF, hGH, insulin, FSH, and PTH.

Nucleic acids or polynucleotides which may be associated with thehydrogel compositions of the invention include naturally occurringnucleic acids, synthetic nucleic acids and nucleic acids preparedgenetic engineering techniques. The nucleic acids may include genomicnucleic acids, such as the genomic DNA or RNA of viral vectors, orfragments of nucleic acids, such as genes or portions thereof includingportions encoding proteins and capable of causing the expression of suchproteins or fragments thereof when introduced into appropriate cells.The nucleic acids which may be associated with the hydrogel compositionsof the invention may be in the form of one or more linear or circularconstructs such as plasmids, cosmids, yeast artificial chromosomes,bacterial artificial chromosomes or they may be in the form of viral orbacterial vectors. Other nucleic acids which may be incorporated fordelivery include ribozymes, antisense molecules and antisense expressingconstructs.

Whether the active agent is incorporated into the hydrogel compositionsby covalent or noncovalent associations, it is possible to modify theirinteractions with the hydrogels by the incorporation of one or morecharged groups into the hydrogel forming components so that they form acharged hydrogel. Alternatively, it is possible to incorporateadditional charged components, such as charged polymers into thehydrogels. Depending on the degree and nature of the charge this willeffect both the capacity of the hydrogel to associate with thebiologically active moiety and the kinetics of its release.

Synthetic polymers, naturally occurring polymers and polymers derivedfrom naturally occurring polymers may also be incorporated into thehydrogels of this invention. Naturally occurring polymers and theirderivatives that can be incorporated into the hydrogel compositions ofthe invention include polysaccharides and glycosaminoglycans, nucleicacids, proteins and peptides. Useful proteins include, but are notlimited to, antibodies, monoclonal antibodies, chimeric antibodies,single chain antibodies, Fab fragments, enzymes, hormones, albumins,immunoglobins, peptide hormones, growth factors, and structural proteinssuch as collagen, fibrin, fibrinogen fibronectin, vitronectin,osteonectin, and laminin. When these polymers also contain functionalgroups that will react with the functional groups of thehydrogel-forming components, or the hydrogels formed therefrom, theywill become linked to the hydrogel composition.

Hydrogel compositions of the invention may be prepared prior to use.Formed hydrogel compositions may optionally be subject to dehydration orlyophilization in order to remove bound water and used as either theintact hydrogel or reduced to powder or particulate form. Hydrogelcompositions of the invention can also be employed without dehydrationor lyophilization as formed objects or maybe incorporated into deliverysystems including without limitation: ocular insert, suppositories,pessaries, transdermal patches, or capsules filled with the hydrogelcompositions.

Regardless of the form of the hydrogel-forming composition or hydrogelcomposition, it is possible to package the compositions in single use,multiple use or bulk containers. The preparations can optionally besterilized by art-recognized procedures. In one preferred embodiment,the materials are packaged in sterile, single-use containers. In otherembodiments, the materials are packaged for ease of reconstitution byaddition of water, aqueous solutions or suspensions in single ormultiple use containers. In another embodiment, the materials are soldas a kit with a base to initiate gel formation. Optionally the kit cancontain a premeasured amount of water stored in a separate containerand/or directions for preparing the hydrogel.

Hydrogel Formation and Reaction Conditions

In yet another aspect of the invention, methods for forming hydrogelcompositions (i.e., crosslinked polymer compositions capable of forminga hydrogel upon exposure to an aqueous environment) fromhydrogel-forming compositions are provided. Preferably, the hydrogelforming-compositions comprise thiosulfonate hydrogel-forming components,and more preferably thiosulfonate polymer derivatives. It has beenunexpectedly discovered that hydrogel compositions can be formed frommulti-arm thiosulfonate esters of water-soluble polymers without theaddition of a second hydrogel-forming component, such as a crosslinkingagent or redox catalyst, or radiation, such as UV or microwave. Due tothese mild gelling conditions, the invention is compatible for use invariety of sensitive applications, including biological applications.Further, it was unexpectedly discovered that the physical properties ofthe hydrogel can be adjusted to desired parameters by controlling thegelling conditions.

In general, methods of the invention include providing ahydrogel-forming composition comprising at least one thiosulfonatepolymer derivative, and exposing the composition to a basic pH underdesired gelling conditions to thereby initiate crosslinking. Thehydrogel-forming composition will preferably be substantially free of asecond hydrogel-forming component such as a crosslinking agent or redoxcatalyst. Further, the thiosulfonate polymer derivative of thehydrogel-forming composition is preferably a multi-arm thiosulfonateester of a water-soluble polymer, such as those of Formulas I-IIIdescribed above. In one embodiment, the hydrogel-forming composition ispreferably a single component hydrogel-forming composition as describedabove. Alternatively, the hydrogel-forming composition can comprise amixture of different thiosulfonate polymer derivatives.

Again, without intending to be limited by theory, it is believed thatthe thiosulfonate polymer derivatives of the invention release asulfonate leaving group upon exposure to a base, thereby generating areactive thiol group in situ. This reactive thiol group is thenavailable for reaction with appropriate electrophilic functional groupssuch as thiosulfonate groups. As such, upon exposure to a base, thethiosulfonate hydrogel polymer derivatives of the invention release asulfonate leaving group to generate in situ a thiol group, which thenforms a disulfide bond with remaining thiosulfonate groups of otherthiosulfate polymer derivatives to thereby form a crosslinked hydrogelcomposition.

More particularly, it was found that the physical properties of ahydrogel formed according to the method of the invention can becontrolled through the selection of desired gelling conditions such aspH, temperature, and polymer concentration. These gelling conditions caninfluence the gel time, viscosity, and degree of crosslinking within thehydrogel. As such, according to a preferred embodiment of the invention,a hydrogel with specific desired physical properties can be formed froma single component hydrogel-forming composition of thiosulfonate estersof water-soluble polymers without the need for a second hydrogel-formingcomponent or exposure radiation. The hydrogel so formed can exhibitdesired physical properties and be substantially free from by-productsof a second hydrogel-forming component.

Preferred gelling conditions include any basic pH and/or temperaturethat does not cause undesired degradation of the hydrogel-formingcomposition. Such conditions can be determined experimentally. PreferredpH values range from about 7.4 to about 11, more preferably from about7.4 to about 9.0, with a pH of about 8.0 being particularly preferred.Preferred temperatures range from about 20° C. to about 50° C., withtemperatures ranging from about 25° C. to about 37° C. beingparticularly preferred. Preferred thiosulfonate polymer derivativeconcentrations are generally only limited by solubility, but preferablyrange from about 2% to about 25% wt/vol, and more preferably from about2% to about 10% wt/vol, based on the total volume of hydrogel-formingcomposition under basic conditions. Although any base can be used toinitiate the reactions required for crosslinking, exemplary bases suitedfor this purpose include sodium hydroxide, sodium acetate, ammoniumhydroxide, potassium hydroxide, ammonium acetate, potassium acetate,sodium phosphate, potassium phosphate, sodium citrate, sodium formate,sodium sulfate, potassium sulfate, potassium fumerate, and combinationsthereof.

The gelling conditions may be selected to obtain a desired gel time,hydrogel viscosity, and degree of crosslinking, as recognized by oneskilled in the art. Such gelling conditions can result in a gel timeranging from about 1 minute (or less) to about 10 hours or longer. Asused herein, gel time generally refers to the time until no visible ormacro-flow of the hydrogel-forming composition is evident followingexposure to basic conditions.

The hydrogel-forming composition may be an aqueous solution orsuspension comprising the hydrogel-forming components (e.g., athiosulfonate polymer derivative). The hydrogel-forming components canalso be suspended in nonaqueous fluid carriers, including, withoutlimitation, hyaluronic acid, dextran sulfate, dextran, succinylatednoncrosslinked collagen, methylated noncrosslinked collagen, glycogen,dimethylsulfoxide, glycerol, dextrose, maltose, triglycerides of fattyacids (such as corn oil, soybean oil, and sesame oil), and egg yolkphospholipid. Alternatively, the hydrogel-forming composition cancomprise hydrogel-forming components that are in dry form such as coarseor fine powders. Dry formulations of the hydrogel-forming components canbe optionally be shaped, or molded into a variety of forms, includingtablets, capsules or sheets.

In one embodiment, the hydrogel-forming composition can be prepared as amildly buffered solution or suspension at a pH and temperature thatinhibits gel formation. Upon exposure to a basic environment, thecomposition undergoes a change in pH sufficient to permit crosslinkingand hydrogel formation. In another embodiment, the hydrogel-formingcomposition can be in the form of a dry powder that is dissolved in asuitable buffer at a suitable concentration, temperature, and pH toinitiate crosslinking and hydrogel formation. As recognized by one ofordinary skill in the art, such buffers include any known buffer inwhich the thiosulfonate polymer derivative is soluble and the desired pHis achievable, including but not limited to sodium phosphate, PBSbuffer, and borate buffer.

In another embodiment, the hydrogel-forming composition can be anunbuffered or substantively unbuffered solution or suspension ofthiosulfonate polymer derivatives at a concentration and pH that willsubstantially inhibit hydrogel formation. Hydrogel formation can then beinitiated by the addition of an aliquot of pH-adjusting reagent at adesired temperature. In a preferred embodiment, a kit can be providedcomprising (in addition to a gel-forming composition) a separatepredetermined aliquot of a reagent, suspension or solid formulationready for admixture with the hydrogel forming composition.

Biological Applications

The hydrogel compositions of this invention are useful in a variety ofapplications, and are particularly suited for biological applications,including drug delivery, tissue engineering, device coatings, and woundclosure.

Delivery Vehicle

In one embodiment, as discussed above, hydrogel compositions of theinvention can be employed to deliver agents such as active agents.Active agents can be noncovalently associated with the hydrogelcompositions by exposing the hydrogel forming components to the activeagent during gel formation, or by subsequently contacting a hydrogelwith the active agent. Active agents can also be covalently incorporatedinto the hydrogels either through direct linkage to the hydrogel-formingcomponents, or indirectly through a crosslinking agent. Direct andindirect linkages may be formed through, for example, interactions withone or more sulfhydryl groups of the gel, reactive groups on thewater-soluble polymer, or at a terminus thereof. In a preferredembodiment, active agents are attached to the water-soluble polymer ofthe hydrogel-forming components by way of a hydrolytically orenzymatically unstable linkage.

Active agents can also be incorporated into the hydrogel-formingcomponents and hydrogel compositions of the invention through acombination of covalent and noncovalent interactions. In one embodiment,hydrogel-forming components or hydrogel compositions formed therefrommay be covalently linked to a cyclodextrin molecule which provides anon-covalent host for the formation of host guest complexes, and permitsthe association of the biologically active moieties through acombination of covalent and noncovalent interactions. In such anembodiment, the cyclodextrin molecules may be covalently attached to thehydrogel composition through a disulfide linkage formed between thecyclodextrin and the hydrogel forming components.

The hydrogels of this invention may also be used to deliver a broadvariety of diagnostic agents such as radiologically opaque compounds anddyes. The hydrogel compositions of the present invention can also beused to deliver living cells to a desired site of administration. Thedelivered cells may be used for a variety of purposes including therelease hormones and growth factors or the formation of new tissue. Inorder to entrap the cells within a formed hydrogel, the cells can besuspended in a suitable medium and then mixed with the hydrogel-formingcomponents. The hydrogel-forming components can be added in variety ofways including but not limited to addition as: a dry powder or as areconstituted solution which has not yet gelled. Alternatively, thehydrogel-forming components can be prepared as a mildly bufferedsolution or suspension at a pH that inhibits gel formation, and thecells are prepared in a buffered medium sufficient to maintain theviability of the cells upon admixture with the hydrogel-formingcomponents. Formation of the hydrogel at the pH of the resultantcombined hydrogel-forming components and cell culture medium will resultin the entrapping the cells within the hydrogel composition.

Regardless of the specific composition employed, when used as a deliverymeans, the hydrogels and hydrogel-forming compositions of this inventionprovide a depot for sustained release of the materials, such as activeagents, associated with or entrapped within the hydrogels, such thatthey may be released with a simple kinetic profile. Alternatively, thehydrogels can be prepared such that they release materials with two ormore kinetic profiles, which may be accomplished by a variety of means.One approach is to employ two or more different hydrogel-formingcompositions or two or more different formed hydrogels, each providing adifferent rate of release. Another approach includes providing aninitial loading dose entrapped within the hydrogel in addition to a dosecovalently or ionically associated with the gel, such that the materialsassociated with the hydrogel will be released over a longer time period.Still another approach by which the release profile can be controlled isto vary the component from which one or more of the hydrogels employedis formed, such that the hydrogel is degraded at different rates. It isalso possible to employ combinations of the above-mentioned approachesfor providing controlling release.

The hydrogels and hydrogel-forming compositions of this invention can bedelivered to a patient by a variety of means including but not limitedto subdermally (subcutaneously), orally, intravenously,intraperitoneally, dermally (transdermal delivery), intradermally,intratumorily, intraocularly, intravicscerally, intraglandularly,intravaginally, intrastromally, intrasynovially intrasinus,intraventricullarly, intrathecally, intramuscularly, and intrarectally.The hydrogel compositions of the invention can also be applied tosurgical sites (before, during, or after surgery, or a combinationthereof). It will be understood that certain sites are selected toachieve systemic distribution of materials and others, such asintraocular, intrathecal and intratumor are selected for targeted (i.e.,local) delivery of materials.

Tissue Engineering

The hydrogel compositions of the invention can also be used for tissueengineering purposes, such as the formation of scaffold materials andtissue augmentation. Examples of soft tissue augmentation includesphincter (e.g., urinary, anal, esophageal) augmentation and thetreatment of rhytids and scars. Examples of hard tissue augmentationapplications include the repair and/or replacement of bone and/orcartilaginous tissue. The hydrogel composition can also be formed as ascaffold that provides a defined surface area for tissue growth.

A general method for effecting augmentation of tissue involves injectingsolutions or suspensions of hydrogel-forming components within thetissue in need of augmentation. The pH of the hydrogel formingcomponents may be adjusted through use of appropriate physiologicallycompatible buffer solutions. Generally a small gauge needle can beemployed to minimize tissue damage. Once delivered, the composition willform the hydrogel in situ. When the hydrogel-forming compositioncontains bioactive agents such as collagen, the collagen can beincorporated into the matrix by reacting with the hydrogel-formingcomponents as discussed above. Groups present on the hydrogel-formingcomponents may also interact with groups present in the patient's tissue(e.g., pendant thiol groups present on the patients tissues) resultingin the attachment of the formed hydrogel to the tissues.

Vascular Occlusion

Formed hydrogel compositions that have been molded into the form of atube, string or coil, which have been subject to dehydration orlyophilization can be surgically implanted or delivered via catheter toa site of vascular damage or malformation. Hydrogel compositions thathave been subject to dehydration or lyophilization compact in size andwill rehydrate inside the vessel, swelling to their original shape. Thehydrogels of this invention can thus be employed to reinforce damagedvessel tissue as occurs for example in an aneurysm, or for the purposeof treating vascular occlusion.

Bioadhesive Applications and Wound Closure

In one preferred embodiment of this invention, the compositions of theinvention can be prepared such that they are particularly suitable foruse as bioadhesives, for example, for use in surgery and for woundclosure. Such compositions are suitable for forming an attachmentbetween the surfaces of two tissues, or between a native tissue surfaceand a normative tissue surface or a surface of a synthetic implant.

Generally, the compositions containing hydrogel forming components areapplied to the surfaces of the tissues to be joined either as a dryformulation such as a powder or a thin sheet, or as a solution ofhydrogel-forming components that may be applied by injecting, sprayingor brushing. The tissues are then joined and held in place until thehydrogel is formed in situ. As the gel forms, it may react with pendantreactive groups present on the tissue surface to form covalent bondsthat aid in anchoring the faces of the tissue to be joined.

Hydrogel formation will generally be complete by one hour but can becomplete in substantially less time, such as from about 5 to about 10minutes. The time required for joining the tissue will be affected by anumber of variables including the type and concentration of thehydrogel-forming components employed, the presence of additionalcomponents such as collagen. It is possible to reduce the time requiredfor hydrogel formation by either increasing the concentration of thehydrogel-forming compositions or adjusting the pH. Application ofhydrogel-forming compositions in dry form will result in slower hydrogelformation as the materials will require rehydration.

Implant Coatings

Another use of the hydrogel compositions of the invention is as abiocompatible coating material on a device intended for implantationinto the body (referred herein as implants). Implants include, withoutlimitation, artificial blood vessels, heart valves, artificial organs,bone prostheses, implantable lenticules, vascular grafts, stents,stent/graft combinations, and so on.

For coating implants, solutions containing hydrogel-forming componentscan be applied to the surface of the implant by any convenient meansincluding, but not limited to, brushing, dipping or spraying. Theimplant can be applied to the target tissue prior to hydrogel formationor after hydrogel formation. As discussed above, the hydrogel-formingcomponents may interact with reactive pendant groups on the surface ofthe surrounding tissue and the implant, which will aid in anchoring theimplant in position.

While coating the surface of implants will generally aid in anchoringthe implant, coating may also provide additional benefits such reducedthrombogenicity, which is an important consideration in applicationssuch as artificial blood vessels and heart valves, vascular grafts,vascular stents, and stent/graft combinations. The hydrogel-formingcompositions may also be used to coat implantable surgical membranes(e.g., monofilament polypropylene) or meshes (e.g., for use in herniarepair).

Ophthalmic Applications

Because of their optical clarity, the hydrogel compositions of theinvention are particularly well suited for use in ophthalmicapplications, including gel-based eye drops for delivering activeagents.

Prevention of Tissue Adhesions

Another use of the hydrogel compositions of the invention is to coattissues in order to prevent the formation of adhesions following surgeryor injury to internal tissues or organs. It may also be desirable toincorporate proteins such as albumin, fibrin or fibrinogen into thehydrogel composition to promote cellular adhesion.

In a general method for coating tissues to prevent the formation ofadhesions following surgery, a thin layer of the hydrogel-formingcomponents is applied to the tissues before substantial crosslinking hasoccurred. Application of the hydrogel-forming components to the tissuesite may be by extrusion, brushing, spraying (as described above), or byany other convenient means. Following application of thehydrogel-forming components to the surgical site, crosslinking isinitiated and allowed to continue in situ prior to closure of thesurgical incision. Once crosslinking has reached equilibrium, tissueswhich are brought into contact with the coated tissues will not stick tothe coated tissues. At this point in time, the surgical site can beclosed using conventional means (sutures, and so forth).

In general, compositions that exhibit a relatively short gel time (i.e.,5-15 minutes following exposure to a basic environment) are preferredfor use in the prevention of surgical adhesions, so that the surgicalsite may be closed relatively soon after completion of the surgicalprocedure.

All articles, books, patents and other publications referenced hereinare hereby incorporated by reference in their entireties.

EXAMPLES

The preparation of hydrogel-forming components and the formation ofhydrogel compositions comprising these components may be accomplished bya variety of means including those outlined below. However, theembodiments exemplified below are in no way to be considered as limitingthe scope of the invention.

Unless otherwise indicated, all PEG reagents are available from NektarTherapeutics, Huntsville, Ala. All other reagents are available fromcommercial suppliers such as Aldrich, St. Louis, Mo.

Example 1 Synthesis of 4-Arm PEG 10 kDa Methanethiosulfonate andHydrogel Formation

Sodium Methanethiosulfonate

Sodium methane sulfinate (3.0 g) and sulfur (1.41 g) in methanol (180ml) were refluxed under argon for 45 minutes. The mixture was cooled toroom temperature, filtered, and the filtrate evaporated to dryness underreduced pressure to obtain the product (3.2 g). NMR (DMSO-d6): 2.97 ppm(s, CH₃SO₂SNa), peak at 1.82 ppm (s, CH₃SO₂Na) absent.

Schematically, the reaction can be represented as follows:

4-Arm PEG 10 kDa Methanesulfonate

4-arm PEG 10 kDa, (MW 10,000; 20.0 g; 8 mmol of hydroxyl groups) (NOFCorporation, Tokyo, Japan/Nektar Therapeutics, Huntsville, Ala.) wasdried by azeotropic distillation from 400 ml of chloroform. Chloroform(300 ml) was added to dissolve the residual syrup and the solution wascooled to 4° C. The flask was purged with dry argon and triethylamine(“TEA” 2.01 ml, 14.4 mmol) was injected followed by slow injection ofmethanesulfonyl chloride (1.02 ml, 13.2 mmol). The reaction mixture wasstirred overnight under argon while the bath rose to ambienttemperature. Anhydrous ethanol (5 ml) was added and the mixture stirredat room temperature for 30 minutes. Anhydrous sodium carbonate (10.0 g)was added to the reaction mixture and the resulting solution stirred atroom temperature for one hour at which point it was filtered. Thefiltrate was concentrated to dryness, and 400 ml of 2-propanol wasadded. The precipitated product was collected by filtration and driedunder vacuum. Yield: 20.0 g. NMR (DMSO-d6): 3.51 ppm (s, PEG backbone),4.31 ppm (t, —CH₂OSO₂—). Integration indicated 95.3% substitution bymethanesulfonate.

Schematically, the reaction can be represented as follows:

4-Arm PEG 10 kDa-Methanethiosulfonate

4-arm PEG 10 kDa methanesulfonate (5.0 gm, 2.0 mmoles) was dried byazeotropic distillation. Anhydrous ethanol (80 ml) was added to theresidual syrup. The flask was purged with dry argon. Sodiummethanethiosulfonate (1.07 gm, 90%, 7.2 mmoles) was added under argonand the mixture refluxed under argon overnight. The mixture was filteredand the filtrate concentrated on a rotary evaporator at 40° C. untildry, followed by addition of 100 ml 2-propanol. The precipitated productwas collected by filtration and dried under high vacuum overnight. Thedried product (4.0 g) was dissolved in 100 ml of dichloromethane andwashed with 100 ml of sodium phosphate buffer pH. 5.0 (10% w/vNaH₂PO₄—Na₂HPO₄). The aqueous solution was back extracted with threealiquots of dichloromethane (200 ml). The combined dichloromethanesolutions were dried over anhydrous sodium sulfate. The mixture wasfiltered and the filtrate concentrated to near dryness on a rotaryevaporator. The product was precipitated by the addition of 100 ml of2-propanol and 100 ml ethyl ether. The precipitated product wascollected by filtration and then dried under vacuum. Yield 3.1 g. NMR(DMSO-d6): 3.51 ppm (s, PEG backbone). The complete disappearance ofpeak at 4.31 ppm (t, —CH₂OSO₂—) indicated all mesylate groups had beensubstituted. The peak for the CH₃SO₂S— methyl group is under PEGbackbone peak. Free sodium methanethiosulfonate was effectively removedbased upon the absence of a peak at (2.94 ppm, s).

Schematically, the reaction can be represented as follows:

Hydrogel formation from 4-arm PEG 10 kDa-methanethiosulfonate.

A 5% w/v solution of 4-arm PEG 10 kDa methanethiosulfonate was preparedin 100 mM sodium phosphate buffer (pH 8). The solution formed a hydrogelin about three hours at 23° C. and in about one hour at 37° C.

Schematically, the reaction can be represented as follows:

Example 2 Synthesis of 4-Arm PEG 10 kDa p-Toluenethiosulfonate andHydrogel Formation

4-Arm PEG 10 kDa Methanesulfonate

4-arm PEG 10 kDa, (20.0 g, 8 mmol of hydroxyl groups) (NOF Corporation,Tokyo, Japan) was dissolved in 400 ml of chloroform and evaporated todryness under vacuum at 40° C. The residue was dissolved in 300 mlchloroform and the flask was purged with dry argon. Triethylamine(“TEA,” 2.01 ml, 14.4 mmol) was added under argon. Methanesulfonylchloride (1.02 ml, 13.2 mmol) was slowly added and the reaction mixturewas stirred overnight under argon while the bath temperature rose toroom temperature. Anhydrous ethanol (5 ml) was added and the mixturestirred at room temperature for 30 minutes. Sodium carbonate (10.0 g)was added and the solution was stirred at room temperature for one hour,after which the mixture was filtered. The filtrate was concentrated todryness, followed by addition of 400 ml of 2-propanol. The precipitatedproduct was collected by filtration and dried under vacuum. Yield 20.0g. NMR (DMSO-d6): 3.51 ppm (s, PEG backbone), 4.31 ppm (t, —CH₂OSO₂—).Integration indicated 95% substitution by methanesulfonate.

Schematically, the reaction can be represented as follows:

4-Arm PEG 10 kDa p-Toluenethiosulfonate

4-arm PEG 10 kDa methanesulfonate, (10.0 gm, 4.0 mmoles) in chloroform(100 ml) was evaporated to dryness on rotary evaporator at 40° C. Theflask was purged with dry argon and 150 ml of anhydrous ethanol wasadded to the remaining syrup followed by addition of 3.734 g (16.0mmoles) of potassium p-toluenethiosulfonate. The mixture was refluxedunder argon overnight and the solvent removed under vacuum. The productwas dissolved in 500 ml of 1M NaH₂PO₄—Na₂HPO₄ buffer solution pH 5.8,containing 10 wt % NaCl, and the aqueous solution was extracted threetimes with 200 ml aliquots of dichloromethane. The combineddichloromethane extracts were dried over anhydrous sodium sulfateevaporated to dryness, and the product precipitated with 100 ml of2-propanol and 100 ml of ethyl ether. The product was collected byfiltration and dried under vacuum. Yield 8.1 g. NMR (DMSO-d6): 1.37 ppm(s, —OC(CH₃)₃), 2.43 ppm (s, CH₃—CH₂═CH₂/Ar), 3.51 ppm (s, PEGbackbone), 6.76 ppm (t, —CH₂NH—CO—), 7.49 ppm (dd, CH₃—CH₂═CH₂/Ar), 7.82ppm (dd, CH₃—CH₂═CH₂/Ar). Based upon integration, the product was 95%substituted.

Schematically, the reaction can be represented as follows:

Hydrogel Formation

A 5% w/v solution of 4-arm PEG 10 kDa p-toluenethiosulfonate wasprepared in 100 mM sodium phosphate buffer (pH 8). The solution formed ahydrogel in about three hours at 23° C. and in about one hour at 37° C.

Schematically, the reaction can be represented as follows:

Example 3 Synthesis of 4-Arm PEG (2-Amidoethyl)Methanethiosulfonate andHydrogel Formation

Synthesis of 4-Arm PEG (2-amidoethyl)methanethiosulfonate

4-arm PEG 10 kDa 1-benzotriazolyl carbonate (4-arm PEG BTC) (4.0 g, 1.6mmol of —BTC) available from Shearwater Corporation was dissolved in 40ml of anhydrous acetonitrile. Tetraethyl ammonium acetate (“TEA,” 0.67ml, 4.8 mmol) was added, followed by addition of2-(aminoethyl)methanethiosulfonate HBr salt (0.378 gm, 1.6 mmol.,Toronto Research Chemicals). The reaction mixture was stirred at roomtemperature overnight and the solvent evaporated under vacuum. Theproduct was precipitated by the addition of 2-propanol (100 ml) withvigorous stirring. The product was collected by filtration, rinsed withether, and dried under vacuum. Yield: 3.7 g. NMR (DMSO-d6): 4.07 ppm (t,—CH₂C(═O)N—), 3.51 ppm (s, PEG backbone), 7.54 ppm (—C(═O)NH—). Peak forthe CH₃SO₂S— methyl group is under the PEG backbone peak. Based uponintegration, the product was 99% substituted.

Schematically, the reaction can be represented as follows:

Hydrogel Formation

A 5% w/v solution of 4-arm PEG (2-amidoethyl)methanethiosulfonate wasprepared in 100 mM sodium phosphate buffer (pH 8). The solution formed ahydrogel in about three hours at 23° C. and in about one hour at 37° C.

Schematically, the reaction can be represented as follows:

Example 4 Synthesis of 4-ArmPEG-Glutaryl-(2-Amidoethyl)Methanethiosulfonate and Hydrogel Formation

Synthesis of 4-Arm PEG-Glutaryl-(2-Amidoethyl)methanethiosulfonate

To 1-benzotriazolyl 4-arm PEG glutarate (MW 10,000 DA, 5.0 g, MW 10 kDa,2.0 mmol benzotriazolyl group, Nektar Therapeutics, Huntsville, Ala.) inanhydrous chloroform (60 ml) was added methyl2-amino-1-ethanethiosulfonate (0.472 gm, 2.0 mmol) followed bytriethylamine (“TEA,” 0.84 ml, 6.0 mmole) and the mixture was stirredovernight. The solvent was removed by evaporation under vacuum and theproduct precipitated by addition of 2-propanol with vigorous stirring.The product was collected by filtration, washed with ethyl ether, anddried under vacuum.

Yield: 4.7 g. NMR (DMSO-d6): 8.12 ppm (—C(═O)NH—), 4.12 ppm (t,—CH₂—O—C(═O)—), 3.51 ppm (s, PEG backbone), 2.31 ppm (t, —O—C(═O)CH₂—),2.12 ppm (t, —CH2C(═O)N—), 1.74 ppm (m, —(O═C)—C—CH2—C—C(═O)—). 90%substitution was determined.

Schematically, the reaction can be represented as follows:

Hydrogel Formation

A 5% solution of 4-arm PEG-glutaryl-(2-amidoethyl)methanethiosulfonatewas dissolved in 100 mM sodium phosphate buffer (pH 7.4). The solutionformed a hydrogel in about 1 hour at 23° C.

Schematically, the reaction can be represented as follows:

Example 5

Formation of Hydrogel From 4-Arm 10 kDa PEG Methanethiosulfonate and4-Arm 10 kDa PEG Thiol

A 5% wt./vol. solution (5 ml) of 4-arm 10 kDa PEG methanethiosulfonatein 0.1 M sodium phosphate buffer and a 5% wt./vol. solution (5 ml) of4-arm 10 kDa PEG thiol were mixed with shaking at room temperature. Ahydrogel formed in less than one minute.

Schematically, the reaction can be represented as follows:

Example 6 Effects of Buffer pH, Temperature and Polymer Wt % on Gel Time

Several gelation conditions, including temperature, the weightpercentage of components which will form the hydrogel (polymer weightconcentration), pH of buffer solution, affect the rate of hydrogelformation. The effects of variables on gelation time were observed inthe following experiments.

Sodium phosphate buffers (0.1 M pH at 7.00, 7.50, 8.00 respectively)were used to dissolve 4-arm PEG10 kDa-methanethiosulfonate (NektarTherapeutics, Huntsville, Ala.) in the glass vials at the concentration(% wt/vol) listed in Table 1. Each vial contained 1000 mg mixtures of 4arm PEG 10 kDa methanethiosulfonate and buffer. Each vial was shakenwhile dissolving and was then incubated without agitation at roomtemperature (24° C.) or in an incubator (37.0° C.) as recorded in thefollowing table. Gel time was recorded for each vial. When the vial wasturned upside down, the gel was considered formed if the material didnot begin flowing immediately.

Results are detailed in Table 1, below.

TABLE 1 Effects of Polymer Concentration, Buffer Solution pH andIncubating Temperature on Gel Time Incubating Vial Gel Time Conc. 0.1MSodium Temperature No. (min) (% wt/vol) phosphate buffer pH (° C.) 1 5852% 7.50 24 2 259 2% 8.00 24 3 205 2% 7.50 37 4 81 2% 8.00 37 5 682 5%7.00 24 6 230 5% 7.50 24 7 103 5% 8.00 24 8 175 5% 7.00 37 9 70 5% 7.5037 10 40 5% 8.00 37 11 586 10% 7.00 24 12 210 10% 7.50 24 13 103 10%8.00 24 14 155 10% 7.00 37 15 65 10% 7.50 37 16 35 10% 8.00 37

Many modifications and other embodiments of the invention will come tomind to one skilled in the art to which this invention pertains havingthe benefit of teachings presented in the foregoing descriptions and theassociated drawings. Therefore, it is to be understood that theinvention is not to be limited to the specific embodiments disclosed andthat modifications and other embodiments are intended to be includedwithin the scope of the appended claims. Although specific terms areemployed herein, they are used in a generic and descriptive sense onlyand not for purposes of limitation.

1. A compound having the formula:

wherein POLY is a water-soluble polymer, (n) is 3 to about 25, X is alinking group, Y is a moiety derived from a molecule having at leastthree nucleophilic groups, and R is an organic radical.
 2. The compoundof claim 1, wherein the water-soluble polymer is a poly(ethyleneglycol).
 3. The compound of claim 1, wherein (n) is 3 to
 10. 4. Thecompound of claim 3, wherein (n) is
 3. 5. The compound of claim 3,wherein (n) is
 4. 6. The compound of claim 3, wherein (n) is
 5. 7. Thecompound of claim 6, wherein (n) is
 6. 8. The compound of claim 1,wherein the organic radical is alkyl or aryl.
 9. The compound of claim1, wherein the organic radical is methyl.
 10. The compound of claim 1,wherein the organic radical is phenyl.
 11. The compound of claim 2,wherein the poly(ethylene glycol) has a molecular weight of from 200 Dato about 20,000 Daltons.
 12. The compound of claim 11, wherein thepoly(ethylene glycol) has a molecular weight of from 500 Da to about15,000 Daltons.
 13. The compound of claim 11, wherein the poly(ethyleneglycol) has a molecular weight of from 600 Da to about 6,000 Daltons.14. The compound of claim 1, wherein the molecule having at least threenucleophilic groups is selected from the group consisting of glycerol,oligoglycerols, pentaerythritol, carbohydrates, and cyclodextrin. 15.The compound of claim 1, wherein the molecule having at least threenucleophilic groups is glycerol.
 16. The compound of claim 1, whereinthe molecule having at least three nucleophilic groups is anoligoglycerol.
 17. The compound of claim 1, wherein the molecule havingat least three nucleophilic groups is


18. The compound of claim 1, wherein the molecule having at least threenucleophilic groups is pentaerythritol.
 19. The compound of claim 1,wherein the molecule having at least three nucleophilic groups is1,2,3-propane-triamine.